WO2019055829A1 - Methods for detecting cancer biomarkers - Google Patents

Abstract

Circulating tumor biomarkers and methods of use thereof are provided.

Description

METHODS FOR DETECTING CANCER BIOMARKERS

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application 62/559,049, entitled "Circulating Tumor Biomarkers and Methods of Use Thereof, filed September 15, 2017. The contents of the aforementioned application is hereby incorporated by reference herein in its entirety.

FIELD

The disclosure relates generally to detecting mutations in cancers of patients receiving cancer therapy and more particularly to detecting genetic mutations in brain cancers, including pediatric brain cancers.

BACKGROUND

Pediatric brain cancers are the leading cause of cancer related mortality in children under

14 years of age¹. Among these, mahgnant tumors forming in midline anatomical brain structures (e.g. pons, thalamus, spinal cord) have dismal outcomes². Children diagnosed with midline gliomas (MLGs) - including children with diffuse intrinsic pontine glioma (DIPG) - often die within a year of diagnosis.

MLGs harbor mutations in genes encoding canonical histones H3.1 (HIST1H3B/C), H3.2

{HIST2H3C), and non-canonical histone H3.3 (H3F3A) variants²⁴. Thus, these tumors were recently re-classified by the World Health Organization (WHO) as diffuse midline glioma, H3 K27M-mutant⁵. While surgical biopsies for diagnosing brainstem tumors have proven safe, characterize tumor biology and potentially guide therapy^6"7 the sensitive anatomical location of these tumors and the need for specialized expertise has prohibited a wider application of surgical biopsies, especially when performing biopsies for tumor recurrence. MR imaging combined with clinical examination is currently being used to assess therapy responses, both of which at times lack sensitivity and specificity.^8"10 As new biologically targeted strategies, specifically immunotherapy approaches are entering the clinic for children with MLGs, the inability to accurately assess disease response, and treatment related molecular changes remain significant challenges.^11"12 Despite ongoing biopsy- informed clinical trials, including convection-enhanced delivery (CED) of drugs, monitoring tumor response to treatment remains a challenge. Magnetic resonance imaging (MRI) is not a sensitive method for monitoring tumor burden and is often slow to reflect tumor progression and regression.

Therefore, there is a need for the development of safe and sensitive tests of cancer biomarkers for the brain.

SUMMARY

The disclosure provides new methods for diagnosing cancers, including pediatric brain tumors.

Accordingly, in one aspect, the disclosure provides a method of determining the efficacy of a cancer therapy, said method comprising:

a) administering a cancer therapy to a subject in need thereof;

b) obtaining a biological sample from the subject after administration of the cancer therapy to the subject; and

c) determining the presence of a mutation in one or more genes selected from the group consisting of H3F3A, HIST1H3B, HIST1H3C, HIST2H3C, ACVR1, PPM ID, PIK3R1, PIK3CA, IDH1, and BRAF in the biological sample,

wherein a decrease in the presence of the mutation(s) compared to the amount observed prior to administration of the cancer therapy indicates that the cancer therapy administered to the subject is effective. In one embodiment, steps b) and c) are repeated at least once, wherein a decrease in the presence of the mutation(s) over time indicates that the cancer therapy administered to the subject is effective. In some embodiments, step c) comprises performing a method selected from the group consisting of quantitative polymerase chain reaction (qPCR), quantitative real-time polymerase chain reaction (qRTPCR), digital droplet PCR, (ddPCR), sequencing, northern blotting, or Southern blotting.

In other aspects, the disclosure provides a method of treating cancer in a subject in need thereof, said method comprising:

a) determining the presence of a mutation in one or more genes selected from the group consisting of H3F3A, HIST1H3B, HIST1H3C, HIST2H3C, ACVR1, PPM ID, PIK3R1, PIK3CA, IDH1, and BRAF in a biological sample, and b) administering a cancer therapy to said subject if the mutation(s) is present. In some embodiments, step a) comprises performing digital PCR or drop digital PCR (ddPCR).

In other aspects, the disclosure provides a method of detecting a mutation in one or more genes selected from the group consisting of H3F3A, HIST1H3B, HIST1H3C, HIST2H3C, ACVRl, PPMID, PIK3R1, PIK3CA, IDHl, and BRAF in a blood or plasma sample from a human subject between 1 and 18 years of age, said method comprising performing digital PCR or droplet digital PCR (ddPCR) on the blood or plasma sample to determine the presence of a mutation in one or more genes selected from the group consisting of H3F3A, HIST1H3B, HIST1H3C, HIST2H3Q ACVRl, PPMID, PIK3R1, PIK3CA, IDHl, and BRAF in a biological sample. In some embodiments the subject has cancer. In one embodiment, the cancer is a brain tumor. In some embodiments, the brain tumor is a diffuse glioma or a diffuse intrinsic potine glioma (DIPG).

In some embodiments, the biological sample is a fluid. In some embodiments, the biological fluid is blood, plasma, serum, CSF, or urine.

In some embodiments, the cancer is a brain tumor. In one embodiment, the cancer is a pediatric brain tumor. In some embodiments, the brain tumor is a diffuse glioma or a diffuse intrinsic pontine glioma (DIPG).

In some embodiments, one or more genes comprise H3F3A. In one embodiment, the mutation is a K27M mutation in H3F3A. In another aspect of this embodiment, of any of the above methods, the method further comprises determining the presence of a mutation in at least one other gene selected from the group consisting of HIST1H3B, HIST1H3C, HIST2H3C, ACVRl, PPMID, PIK3R1, PIK3CA, IDHl, and BRAF. In another embodiment, the mutation is a K27M mutation in H3F3A. In another aspect of this embodiment, of any of the above methods, the method further comprises determining the presence of a mutation in at least one other gene selected from the group consisting of HIST1H3B, ACVRl, PPMID, PIK3R1, and PIK3CA. In another aspect of this embodiment, of any of the above methods, the method further comprises determining the presence of a mutation in at least one other gene selected from the group consisting of HIST1H3B, ACVRl, PPMID, and PIK3R1.

In other aspects, the disclosure provides an oligonucleotide probe comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 19-36, wherein the probe further comprises a label. In another aspect, the disclosure provides a composition comprising at least one oligonucleotide probe comprising one of SEQ ID NOs: 19-36, wherein the probe further comprises a label.

BRIEF DESCRIPTION OF THE FIGURES FIGS. 1A and IB provide an overview of biofluid specimens utilized for detection of ctDNA in diffuse midline glioma patients. FIG. 1A provides demographic, clinical, and molecular information for patients analyzed herein. Genomic alterations were obtained by tumor sequencing data generated for the PNOC003 trial, biorepository sequencing project, or other clinical studies. FIG. IB is a flow chart of biofluid samples analyzed herein. Patients were either enrolled in PNOC003 trial (DIPG) or Children's National (CN) brain tumor biorepository (MLG); *patients enrolled on 1339 to donate specimens at postmortem.

FIGS. 2A-N are graphical representations of genomic DNA validation for specific detection of mutant and wild type alleles of genes encoding driver mutations in pedatric diffuse midline gliomas. Shown are representative ddPCR plots for mutation detection using tumor genomic DNA from seven MLG patients and two non-CNS diseased brain tissue controls. FIGS. 2A-2G are from tumor tissue; FIGS. 2H-2N are from two non-CNS diseased brain tissue controls.

FIG. 3A-G are graphical representations of results obtained when assessing the sensitivity of a ddPCR platform with pre-amplification quantities of 2500 pg, 250 pg, 25 pg, and 2.5 pg of input DNA Genomic DNA. The DNA was obtained from frozen tumor tissue obtained at postmortem, from a H3.3 K27M mutant DIPG patient was diluted and used to assess for sensitivity. FIGS. 3A-3C are samples assessed without pre-amplification; FIGS. 3D-3G are from samples assessed with preamplification.

FIGS. 4A-4B are graphical representations assessing the specificity of s ddPCR platform by testing for H3.3 K27M in H3 wildtype DIPG tumor tissue and liquid bio me. FIG. 4A shows matched tumor tissue and CSF collected at postmortem from the same H3 WT DIPG patient who was negative for H3F3A p.K27M. FIG. 4B shows that plasma collected at initial diagnosis was negative for H3F3A p.K27M in three H3 wild type DIPG patients.

FIG. 5A-B show schematics of biofluid specimen processing for ddPCR analysis. FIG. 5A shows that genomic DNA was isolated from tumor tissue and used for ddPCR, while plasma and CSF were frozen upon collection, processed for ctDNA isolation, preamplified, and analyzed by ddPCR. FIG. 5B shows that circulating DNA isolated from biofluid specimens were analyzed in a three-step method by ddPCR, which includes dropletization (by a'Source' machine), PCR, followed by detection in the 'Sense' machine. An example is shown for H3F3A p.K27M detection, where LNA probes detect a single nucleotide change differentiating mutant and wild type alleles during PCR.

FIGS. 6A-F are graphic and pictorial representations showing detection of an histone 3 p.K27M mutation (H3K27M) ctDNA in biofluids of patients diagnosed with midline glioma. FIG. 6A shows detection of H3K27M ctDNA in CSF collected throughout a disease. FIG. 6B shows significantly higher ctDNA MAFs for H3K27M were detected in CSF compared to plasma. FIG. 6C shows higher H3K27M MAFs were present in CSF compared to plasma in the same patient. FIG. 6D shows that plasma ctDNA collected at diagnosis in DIPG patients enrolled in the PNOC003 clinical trial analyzed for H3K27M. Red asterisk denotes histone 3 wild type DIPGs. FIG. 6E shows that detection of H3K27M plasma ctDNA at post radiation in two patients that lacked detection at diagnosis. FIG. 6F shows an Increase in tumor volume by MRI at post radiation compared to diagnosis in patients shown in FIG. 6C.

FIGS. 7A-F show multiplexed detection of oncohistone and partner mutations in tumor and CSF of MLG patients. Genomic DNA and CSF ctDNA were analyzed from the same MLG patient for multiplexed detection of mutations.

FIGS. 8A-C show CSF collected from various neuroanatomical locations in MLGs. Bar graphs represent average for H3K27M MAF detection from technical duplicates, and error bars represent standard error of mean. FIG. 8A shows ignificantly higher H3K27M MAF was detected in CSF collected from adjacent sites to tumor compared to distant in MLG patients. FIG. 8B shows Cohort based analysis of CSF collected from DIPG patients from different neuroanatomical locations, indicating higher detection in locations closer to brainstem. FIG. 8C shows matched analysis for CSF collected from lateral ventricles and lumbar spine at postmortem from the same DIPG patient, showing similar trends as in (FIG. 8A) and (FIG. 8B).

FIGS. 9A-D show detection of a novel histone 3 mutation in fluid present in a brainstem tumor cyst found in a DIPG patient at postmortem. FIG. 9A is an image of a cyst (indicated by an arrow) in fresh tissue at postmortem. FIG. 9B is an MRI image captured a month before postmortem indicating cyst in pontine tumor. FIGS. 9C-9D show that higher MAF levels were found in cyst fluid (41%) compared to tumor tissue (34%), and CSF (38%) for H3F3A p.K27M. FIGS 10A-B shows detection of H3.3 K27M ctDNA in CSF at postmortem in a DIPG patient that tested negative in CSF collected at diagnosis. FIG. 10A shows detection of

H3K27M ctDNA in CSF collected at postmortem from a patient known to harbor H3.3 K27M in tumor. FIG.10B shows the results of an MRI imaging at diagnosis and postmortem for the same patient CSF was analyzed for in FIG. 10A.

FIGS. 11A-F show that a temporal analysis of plasma ctDNA matches response to therapy in DIPG patients. FIG. 11 A shows serial plasma ctDNA analysis of changes in H3K27M throughout course of treatment in patients that followed PNOC003 recommended therapy. FIG. 1 IB shows dynamic changes in plasma ctDNA and MRI tumor measurements in one patient in response to therapy. FLAIR and Tl-weighted post- gadolinium MRI images prior to treatment demonstrating a prominent expansile pontine mass and an additional focus within right cerebellar hemisphere that demonstrates evidence of enhancement. After treatment, the pontine and right cerebellar hemisphere lesions decreased in size. FIG. 11C shows fluctuations in plasma ctDNA and MRI tumor volumes during therapy. FIG. 1 ID shows a significant decrease in plasma ctDNA and MRI tumor volumes in response to radiation therapy. FIG. 1 IE shows a decrease in plasma ctDNA and MRI tumor burden from diagnosis to pre-cycle 3 of PNOC003 recommended therapy.

FIGS. 12A-12N show longitudinal changes in plasma ctDNA in association with MR imaging findings and clinical assessments. Each line graph represents plasma ctDNA and MRI changes in an individual patient diagnosed with DIPG. The red line depicts MRI tumor measurements and black line depicts changes in temporal plasma ctDNA. Error bars are standard error of mean for technical triplicates of plasma ctDNA assessed for MAF of H3K27M. The colored legend at the bottom of each figure indicates the time point of plasma ctDNA and MRI assessment during course of disease: green for initial diagnosis/biopsy, grey for during therapy, red for tumor growth, and black for the end of therapy following tumor growth/progression.

FIG. 13A-C are histograms showing bio fluid ctDNA and tumor spread as assessed by MRI, genomic, and/or histological studies. FIG. 13A shows tumor extension beyond site of primary tumor (pons or thalamus) as determined based on MRI obtained prior to postmortem, or molecular and/or histopathology of autopsied whole brain specimens. CSF ctDNA was higher in 18 MLG patients with tumor extension as compared to three MLG patients without tumor spread. Tumor involvement beyond pons was assessed by MRI review of patients enrolled in PNOC003. Plasma ctDNA and MRI collected from DIPG patients were analyzed (FIG. 13B) at initial diagnosis and (FIG. 13C) at time points during the course of disease (initial diagnosis, during therapy, and tumor growth). Unlike in CSF, ctDNA levels in plasma were not higher in DIPG patients with tumor spread.

DETAILED DESCRIPTION OF THE DISCLOSURE

The use of circulating tumor DNA (ctDNA) to monitor disease progression is non/minimally- invasive method that has been increasingly employed for disease monitoring in adult cancers including glioblastoma (GBM), melanoma, lung, breast, and colon cancersi3-n74 ,however such studies have not been applied to the pediatric population. Thus, there is an urgent need for the development of ctDNA assays for clinical applications in pediatric CNS patients.

Described herein is a sensitive, specific, rapid and minimally-invasive method for tumor surveillance of pediatric MLGs by monitoring the liquid biome. In embodiments, we show the detection of mutations associated with pediatric MLGs such as DIPG and thalamic tumors using patients' CSF and plasma. Further, using plasma collected through an ongoing clinical trial (PNOC003; NCT 2274987) conducted by the Pacific Pediatric Neuro-Oncology Consortium (PNOC), we show that mutation allelic frequency (MAF) is readily obtained from ctDNA present in liquid biome, and that MAF correlates with treatment response.

The disclosure provides a robust application of the use of ctDNA for tumor profiling, and assessment of tumor response to therapy in pediatric patients with MLGs. These results show the feasibility of incorporating liquid biopsy as a sensitive and minimally invasive tool to inform clinical management for children with MLGs

The use of circulating tumor DNA (ctDNA) to monitor disease progression is a noninvasive method that has been shown to be successful in adult glioblastoma, lung cancer, melanoma, breast cancer, and colon cancer (Tie et al. (2016) Sci. Transl. Med., 8(346):346ra92; Tsao et al. (2015) Sci. Rep., 5:11198, Garcia-Murillas et al. (2015) Sci Transl Med.,

7(302):302ral33; Schiovan et al. (2015) Sci. Transl. Med., 7(313):313ral82; De Mattos-Arruda et al. (2015) Nat. Commun., 6:8839). Several studies have reported the clinical utility of ctDNA as a biomarker for monitoring changes in tumor burden following therapy, resistance to therapy, and emergence of recurrence in melanoma, breast, lung, colon, ovarian cancer (Murtaza et al. (2013) Nature 497(7447): 108- 12; Tsao et al. (2015) Sci. Rep., 5:11198; Tie et al. (2016) Sci. Transl. Med., 8(346) :346ra92; Garcia-Murillas et al. (2015) Sci Transl Med., 7(302):302ral33). Longitudinal changes in ctDNA are a useful biomarker to monitor tumor progression in adult glioblastoma, and in patients with metastasis to the brain from breast and lung cancer (De Mattos-Arruda et al. (2015) Nat. Commun., 6:8839).

However, the validity of ctDNA as a biomarker in the clinical setting for assessing tumor response, longitudinal assessment of tumor size, tumor metastasis, and prediction of relapse is unknown. Moreover, given that ctDNA is detectable in less than 50% of primary brain tumors (Bettegowda et al. (2014) Sci Transl. Med., 6(224):224ra24) and that allelic frequency of H3F3A is low (Lewis et al. (2013) Science 340(6134): 857-61 ; Bender et al. (2013) Cancer Cell

24(5):660-72), a sensitive approach is needed for detection of such driver mutations in pediatric MLGs.

As described below, ctDNA was molecularly characterized in cerebrospinal fluid (CSF) and plasma, and the clinical utility of ctDNA to monitor tumor burden in pediatric midline gliomas (MLGs) was assessed. The disclosure shows that liquid-biopsy platforms can allow detection of ctDNA to reflect tumor regression or progression following therapeutic intervention.

Digital Droplet PCR

In embodiments, methods are performed using highly sensitive PCR-based sequencing method, such as digital droplet PCR.

As used herein, "digital PGR" refers to PCR wherein the sample is divided into discrete subunits prior to amplification by PGR. For example, the sample may be separated into thousands or millions of partitions, each containing either zero or one (or, at most, a few) template molecules. Fluorescent probes may be used to identify amplified target DNA in the partitions. Samples containing amplified product (e.g., fluorescent) are positive and those without product (e.g., no fluorescence) are negative. The ratio of positives to negatives in each sample can be used for quantification. For example, Poisson statistics can be used to determine the absolute template quantity without the need to consider the number of amplification cycles.

As used herein, "droplet digital PCR" (ddPCR) refers to digital PCR wherein the PCR solution is divided into smaller reactions (e.g., picoliter sized) through a water oil emulsion technique, which are then made to ran PCR individually. Typically, the PCR sample is partitioned into nanoliter-size samples and encapsulated in oil droplets, (see, e.g., Hinson et al. (2011) Anal. Chem., 83:8604-8610; Pinheiro et al. (2012) Anal. Chem.,

84:1003-1011).

The PCR process is well known in the art and includes, for example, methods described in US Patent No. 9,984,201, the contents of which are incorporated by reference herein in their entirety. These methods include, e.g., reverse transcription PCR, ligation mediated PCR, digital PCR (dPCR), or droplet digital PCR (ddPCR). For a review of PCR methods and protocols, see, e.g., Innis et al, eds., PCR Protocols, A Guide to Methods and Application, Academic Press, Inc., San Diego, Calif. 1990; U.S. Pat. No. 4,683,202 (Mullis). PCR reagents and protocols are also available from commercial vendors, such as Roche Molecular Systems.

In some embodiments, PCR is carried out as an automated process with a thermostable enzyme. In this process, the temperature of the reaction mixture is cycled through a denaturing region, a primer annealing region, and an extension reaction region automatically. Machines specifically adapted for this purpose are commercially available.

In some embodiments, amplified sequences are also measured using invasive cleavage reactions such as the Invader™ technology (Zou et al, 2010, Association of Clinical Chemistry (AACC) poster presentation on Jul. 28, 2010, "Sensitive Quantification of Methylated Markers with a Novel Methylation Specific Technology; and U.S. Pat. No. 7,011,944 (Prudent et al)).

Suitable next generation sequencing technologies are widely available. Examples include the 454 Life Sciences platform (Roche, Branford, Conn.) (Margulies et al. 2005 Nature, 437, 376-380); Ulumina's Genome Analyzer, GoldenGate Methylation Assay, or Infinium

Methylation Assays, i.e., Infinium HumanMethylation 27K BeadArray or VeraCode GoldenGate methylation array (Illumina, San Diego, Calif.; Bibkova et al, 2006, Genome Res. 16, 383-393; U.S. Pat. Nos. 6,306,597 and 7,598,035 (Macevicz); U.S. Pat. No. 7,232,656 (Balasubramanian et al.)); QX200.TM. Droplet DigitaLTM. PCR System from Bio-Rad; or DNA Sequencing by Ligation, SOLiD System (Applied Biosystems/Life Technologies; U.S. Pat. Nos. 6,797,470,

7,083,917, 7,166,434, 7,320,865, 7,332,285, 7,364,858, and 7,429,453 (Barany et al); the HeUcos True Single Molecule DNA sequencing technology (Harris et al, 2008 Science, 320, 106-109; U.S. Pat. Nos. 7,037,687 and 7,645,596 (Williams et al); U.S. Pat. No. 7,169,560 (Lapidus et al); U.S. Pat. No. 7,769,400 (Harris)), the single molecule, real-time (SMRT.TM.) technology of Pacific Biosciences, and sequencing (Soni and Meller, 2007, Clin. Chem. 53, 1996-2001);

semiconductor sequencing (Ion Torrent; Personal Genome Machine); DNA nanoball sequencing; sequencing using technology from Dover Systems (Polonator), and technologies that do not require amplification or otherwise transform native DNA prior to sequencing (e.g., Pacific Biosciences and Helicos), such as nanopore-based strategies (e.g., Oxford Nanopore, Genia Technologies, and Nabsys). These systems allow the sequencing of many nucleic acid molecules isolated from a specimen at high orders of multiplexing in a parallel fashion. Each of these platforms allow sequencing of clonally expanded or non-amplified single molecules of nucleic acid fragments. Certain platforms involve, for example, (i) sequencing by ligation of dye- modified probes (including cyclic ligation and cleavage), (ii) pyrosequencing, and (iii) single- molecule sequencing.

In an embodiment, methods are performed using digital droplet PCR (ddPCR). Among various methods of analyzing ctDNA derived from biofluids, digital droplet PCR (ddPCR) is one of the most sensitive methods compared to Sanger sequencing, quantitative PCR, and next generation sequencing techniques (Diaz et al. (2014) J. Clin. Oncol. 32(6):579-86; Baker, M. (2012) Nat Meth., 9:541-544).

After PCR amplification, the nucleic acids may be quantified by counting the sub- samples that contain PCR end-product (positive reactions) and the sub- samples containing no PCR end-product (negative reactions) taking into account the Poisson distribution. Digital PCR (dPCR) is, contrary to conventional PCR, not dependent on the number of amplification cycles performed in order to allow for a determination of the initial sample amount, thus eliminating the reliance on uncertain exponential data to quantify target nucleic acids and providing absolute quantification. In embodiments, "Digital droplet PCR" as used herein relates to a digital PCR method in which the initial sample is sub-divided into several droplets constituting the sub- samples.

Herein, ddPCR was used to detect major mutations in ctDNA of liquid biome specimens and to assess dynamic changes in ctDNA during a disease course. H3 27M partners with at least one additional driver mutation in cell cycle regulatory or growth factor signaling pathways (Nikbakht et al. (2016) Nat. Commun., 7:11185). Here, the presence of H3K27M and partner mutations in ctDNA was identified from liquid biome of MLGs. Significantly, the amount of detectable ctDNA in CSF and plasma was assessed with regard to clinical variables such as response to therapy, MRI tumor measurements, stage of disease, tumor spread, overall survival, and neuroanatomieal location of biofluid collection. More specifically, twenty- seven CSF samples were collected at various stages of disease (upfront, recurrence, and postmortem) from pediatric high grade midline gliomas. Upfront and longitudinal plasma samples (n=68) were collected with each MRI, from 22 diffuse intrinsic pontine glioma (DIPG) patients enrolled in a Phase I clinical trial. For monitoring etDNA, a liquid biopsy assay using a sensitive and robust digital droplet PGR system on CSF and plasma samples was developed. Major DIPG driver mutations including H3F3A (e.g., p. 27M),

HIST1H3B (e.g., p. 27M), ACVR1 (e.g., p.G328V/p.R206H), PPM ID (e.g., p.E525X),

PI 3R1 (e.g., p.K567E), and PI 3CA (e.g., p.H1047R) were detected. Samples can also be analyzed for IDH1 (e.g., p,R132H) and BRAF (e.g., p.V600E) mutations. Specifically, H3K27M was detected in 80% of CSF and upfront plasma samples in patients that harbor the histone 3 mutation in tumor. Higher levels of ctDNA were detected in CSF compared to plasma. Longitudinal analysis of plasma showed a significant decrease (p = 0.005) in ctDNA following radiotherapy, whereas MRI tumor measurements did not show a significant decrease in tumor size (n=15). Furthermore, H3K27M was detected in ctDNA of plasma samples in xenograft models of DIPG. Thus, biofluids from patients and animal models (e.g., urine, blood, serum, plasma, CSF) is a suitable medium for detection and quantification of ctDNA. A liquid biopsy can complement or in some cases eliminate the need for biopsies and inform recurrence and tumor response to treatment.

In accordance with the instant invention, methods of treating cancer in a subject in need thereof are pro vided. In a particular embodiment, the method comprises determining the presence of a mutation in histone H3 (e.g., histone H3.3) and administering a therapy (e.g., administering at least one therapeutic agent to the subject) to the subject if the mutation in histone H3 is present. The method may further comprise the step of obtaining a biological sample from the subject. The biological sample of the methods may be a fluid or liquid such as blood, CSF, plasma, or urine. In a particular embodiment, the biological sample is blood or plasma. In a particular embodiment, the therapy administered to the subject is an epigenetic regulating drug such as, without limitation, SAHA and panobiiiostat.

The cancer detected and treated by the instant methods may be a brain tumor. More specifically, the cancer may be a pediatric brain tumor. In a particular embodiment, the cancer is a glioma, particularly a diffuse glioma or a diffuse intrinsic pontine glioma (DIPG). The methods of the instant invention may comprise detenrdning the presence of a mutation(s) in circulating tumor DNA (ctDNA). In a particular embodiment, determining of the presence of the mutation(s) comprises perforating digital PCR. In a particular

embodiment, the determining of the presence of the mutation comprises performing droplet digital PCR (ddPCR). The digital PCR or ddPCR may be performed with at least one primer comprising any one of SEQ ID NOs: 1-18. The digital PCR or ddPCR may be performed with primers comprising each of SEQ ID NOs: 1-18. The digital PCR or ddPCR may be performed with at least one probe comprising any one of SEQ ID NOs: 19-36. The digital PCR or ddPCR may be performed with probes comprising each of SEQ ID NOs: 19-36.

As stated herein above, the instant methods comprise determining the presence of a mutation in histone H3 (e.g., histone H3.3). In a particular embodiment, the mutation in histone H3 is K27M. In a particular embodiment, the histone H3 is H3F3A. In a particular

embodiment, the method comprises determining the presence of a mutation in H3F3A and at least one other gene (e.g., partner gene). The other gene(s) may be, for example, selected from the group of HIST1H3B (e.g., p.K27M), HIST2H3C (e.g., p.K27M), ACVR1 (e.g., p.G328V/p.R206H), PPM1D (e.g., p.E525X), PIK3R1 (e.g., p.K567E), PIK3CA (e.g., p.H1047R), IDH1 (e.g., p.R132H; isocitrate dehydrogenase (NADP(+)) 1, cytosolic; Gene ID: 3417) and BRAF (e.g., p.V600E; B-Raf proto-oncogene, serine/threonine kinase; Gene ID: 673) (e.g., 1, 2, 3, 4, 5, 6, 7, or all 8 genes are analyzed). In a particular embodiment, the other gene(s) is selected from the group of HIST1H3B (e.g., p.K27M), ACVR1 (e.g.,

p.G328V/p.R206H), PPM1D (e.g. p.E525X), PIK3R1 (e.g., p.K567E), and PIK3CA (e.g., p.H1047R) (e.g., 1, 2, 3, 4, or all 5 genes are analyzed). In a particular embodiment, the other gene(s) is selected from the group of HIST1H3B (e.g., p.K27M), ACVR1 (e.g.,

p.G328V/p.R206H), PPM1D (e.g., p.E525X), and PIK3R1 (e.g., p.K567E) (e.g., 1, 2, 3, or all 4 genes are analyzed). Sequence information for certain of these genes is provided below.

H3 histone family member 3A (H3F3A; SEQ ID NO: 37) Gene ID: 3020, Gen Bank Accession Nos. NM_002107.4 and NP_002098.1 (Note: the Met at position 1 is not included in amino acid numbering)

1 ARTKQTARK STGGKAPRKQ LATKAARKSA PSTGGVKKPH RYRPGTVALR EIRRYQKSTE 61 LLIRKLPFQR LVREIAQDFK TDLRFQSAAI GALQEASEAY LVGLFEDTNL CAIHAKRVTI

121 PKDIQLARR IRGERA Histone cluster 1 H3 family member b (HIST1H3B; SEQ ID NO: 38) Gene ID: 8358, Gen Bank Accession Nos. NM_003537.3 and NP_003528.1 (Note: the Met at position 1 is not included in amino acid numbering).

1 ARTKQTARK STGGKAPRKQ LATKAARKSA PATGGVKKPH RYRPGTVALR EIRRYQKSTE

61 LLIRKLPFQR LVREIAQDFK TDLRFQSSAV ALQEACEAY LVGLFEDTNL CAIHAKRVTI

121 PKDIQLARR IRGERA

Activin A receptor type 1 (ACVRl ; SEQ ID NO: 39) Gene ID: 90, Gen Bank Accession

NMJXJl 105.4 and NP_001096.1

1 MVDGVMILPV LIMIALPSPS MEDEKPKVNP KLYMCVCEGL SCGNEDHCEG QQCFSSLSIN

61 DGFHVYQKGC FQVYEQGKMT CKTPPSPGQA VECCQGDWCN RNITAQLPTK GKSFPGTQNF

121 HLEVGLIILS WFAVCLLAC LLGVALRKFK RRNQERLNPR DVEYGTIEGL ITT VGDSTL

181 ADLLDHSCTS GSGSGLPFLV QRTVARQITL LECVGKGRYG EVWRGSWQGE NVAV IFSSR

241 DEKSWFRETE LYNTVMLRHE NILGFIASDM TSRHSSTQLW LITHYHEMGS LYDYLQLTTL

301 DTVSCLRIVL SIASGLAHLH IEIFGTQGKP AIAHRDLKSK ILVKKNGQC CIADLGLAVM

361 HSQSTNQLDV GNNPRVGTKR YMAPEVLDET IQVDCFDSYK RVDIWAFGLV LWEVARRMVS

421 NGIVEDYKPP FYDWPNDPS FEDMRKWCV DQQRPNIPNR WFSDPTLTSL AKLM ECWYQ

481 NPSARLTALR IKKTLTKIDN SLDKLKTDC

Protein phosphatase, Mg2+/Mn2+ dependent ID (PPM1D; SEQ ID NO: 40) Gene ID: 84, Gen Bank Accession Nos. NM„003620.3 and NPJ303611.1

1 MAGLYSLGVS VFSDQGGRKY MEDVTQIWE PEPTAEEKPS PRRSLSQPLP PRPSPAALPG

61 GEVSGKGPAV AAREARDPLP DAGASPAPSR CCRRRSSVAF FAVCDGHGGR EAAQFAREHL

121 WGFIKKQKGF TSSEPAKVCA AIRKGFLACH LAMWKKLAEW PKTMTGLPST SGTTASWII

181 RGMKMYVAHV GDSGWLGIQ DDPKDDFVRA VEVTQDHKPE LPKERERIEG LGGSVMNKSG

241 VNRWWKRPR LTHNGPVRRS TVIDQIPFLA VARALGDLWS YDFFSGEFW SPEPDTSVHT

301 LDPQKHKYII LGSDGLWNMI PPQDAISMCQ DQEEKKYLMG EHGQSCAKML V RALGRWRQ

361 RMLRADNTSA IVICISPEVD NQGNFTNEDE LYLNLTDSPS Y SQETCVMT PSPCSTPPV

421 SLEEDPWPRV NSKDHIPALV RS AFSENFL EVSAEIAREN VQGWIPS D PEPLEENCAK

481 ALTLRIHDSL SLPIGLVP TNSTNTV DQ K LKMSTPGQ MKAQEIERTP PTNFKRTLEE

541 SNSGPLMKKH RRNGLSRSSG AQPASLPTTS QRKNSVKLTM RRRLRGQKKI GNPLLHQHRK

601 TVCVC

Phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1 ; SEQ ID NO: 41) Gene ID: 529 Gen Bank Accession Nos. NM_181523.2 and NP_852664.1

1 MSAEGYQYRA LYDYKKEREE DIDLHLGDIL TV KGSLVAL GFSDGQEARP EEIGWLNGYN

61 ETTGERGDFP GTYVEYIGRK KISPPTPKPR PPRPLPVAPG SSKTEADVEQ QALTLPDLAE

121 QFAPPDIAPP LLIKLVEAIE KKGLECSTLY RTQSSSNLAE LRQLLDCDTP SVDLEMIDVH

181 VLADAFKRYL LDLPNPVIPA AVYSEMISLA PEVQSSEEYI QLLKKLIRSP SIPHQYWLTL

241 QYLLKHFFKL SQTSSKNLL ARVLSEIFSP MLFRFSAASS DNTENLIKVI EILISTEWNE

301 RQPAPALPPK PPKPTTVAN GMN NMSLQD AEWYWGDISR EEVNEKLRDT ADGTFLVRDA

361 STKMHGDYTL TLRKGGNNKL IKIFHRDGKY GFSDPLTFSS WELINHYR ESLAQY PKL

421 DVKLLYPVSK YQQDQWKED NIEAVGKKLH EYNTQFQE S REYDRLYEEY TRTSQEIQMK

481 RTAIEAFNET IKIFEEQCQT QERYSKEYIE KFKREGNEKE IQRIMHNYDK LKSRISEIID

541 SRRRLEEDLK KQAAEYREID KRMNSI PDL IQLRKTRDQY LMWLTQKGVR QKKLNE LGN 601 ENTEDQYSLV EDDEDLPHHD EKTWNVGSSN K KAENLLRG KRDGTFLVRE SSKQGCYACS

661 WVDGEVKHC VINKTATGYG FAEPYNLYSS LKELVLHYQH TSLVQHNDSL VTLAYPVYA

72 QQRR

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA; SEQ ID NO: 42) Gene ID: 5290, Gen Bank Accession Nos. NM_006218.3 and NP_006209.2

1 MPPRPSSGEL WGIHLMPPRI LVECLLPNGM IVTLECLREA TLITIKHELF KEARKYPLHQ

61 LLQDESSYIF VSVTQEAERE EFFDETRRLC DLRLFQPFLK VIEPVGNREE KILNREIGFA

121 IG PVCEFDM VKDPEVQDFR RNILNVCKEA VDLRDLNSPH SRAMYVYPPN VESSPELPKH

181 IYNKLDKGQI IWIWVIVSP NNDKQKYTLK INHDCVPEQV IAEAIRKKTR SMLLSSEQLK

241 LCVLEYQGKY ILKVCGCDEY FLEKYPLSQY KYIRSCIMLG RMPNLML AK ESLYSQLPMD

301 CFTMPSYSRR ISTATPY NG ETSTKSLWVI NSALRIKILC ATYVNVNIRD IDKIYVRTGI

361 YHGGEPLCDN VNTQRVPCSN PRWNEWLNYD IYIPDLPRAA RLCLSICSVK GRKGAKEEHC

421 PLAWGNINLF DYTDTLVSGK ALNLWPVPH GLEDLLNPIG VTGSNPNKET PCLELEFDWF

481 SSWKFPDMS VIEEHANWSV SREAGFSYSH AGLSNRLARD NELRENDKEQ LKAISTRDPL

541 SEITEQEKDF LWSHRHYCVT IPEILPKLLL SVKWNSRDEV AQMYCLVKDW PPIKPEQAME

601 LLDCNYPDPM VRGFAVRCLE KYLTDDKLSQ YLIQLVQVLK YEQYLDNLLV RFLLKKALTN

661 QRIGHFFFWH LKSEMHNKTV SQRFGLLLES YCRACGMYLK HLNRQVEAME KLINLTDILK

721 QEKKDETQKV Q KFLVEQ R RPDFMDALQG FLSPLNPAHQ LGNLRLEECR IMSSAKRPLW

781 LNWENPDIMS ELLFQNNEII FKNGDDLRQD MLTLQIIRIM ENIWQNQGLD LRMLPYGCLS

841 IGDCVGLIEV VRNSHTIMQI QCKGGLKGAL QFNSHTLHQW LKDKNKGEIY DAAIDLFTRS

901 CAGYCVATFI LGIGDRHNSN IMVKDDGQLF HIDFGHFLDH KKKKFGYKRE RVPFVLTQDF

961 LIVISKGAQE CTKTREFERF QEMCYKAYLA IRQHANLFIN LFS MLGSGM PELQSFDDIA

1021 YIRKTLALDK TEQEALEYFM KQ NDAHHGG WTTKMDWIFH TIKQHALN

In accordance with another aspect of the instant invention, methods of determining the efficacy of a cancer therapy are provided. In a particular embodiment, the method comprises 1) administering a cancer therapy to a subject in need thereof, 2) obtaining a biological sample from the subject after administration of the cancer therapy to the subject, and 3) determining the presence of a mutation in in the biological sample (e.g., in histone H3 (e.g., histone H3.3)). The method may comprise obtaining multiple biological samples over time from the subject after administration of the cancer therapy to the subject. The method may comprise obtaining a biological sample from the subject before administration of the cancer therapy to the subject (e.g., a baseline). A decrease in the presence of the mutation (e.g., in histone H3) over time (e.g., when multiple biological samples are obtained after therapy) or compared to the amount observed prior to therapy, indicates that the cancer therapy administered to the subject is effective (e.g., inhibiting tumor growth).

The biological sample of the methods may be a fluid or liquid such as blood, CSF, plasma, serum, or urine. In a particular embodiment, the biological sample is blood or plasma. The cancer monitored by the instant methods may be a brain tumor. More specifically, the cancer may be a pediatric brain tumor. In a particular embodiment the cancer is a glioma, particularly a diffuse glioma or a diffuse intrinsic pontine glioma (DIPG).

The cancer therapy administered to the subject may comprise any type of therapy. For example, the cancer therapy may comprise the administration of at least one chemotherapeutie agent (e.g., a small molecule). The cancer therapy may include radiation therapy.

The methods of the instant invention may comprise determining the presence of the mutation in circulating tumor DNA (ctDNA). In a particular embodiment, determining of the presence of the mutation comprises performing digital PGR. In a particular embodiment, the determining of the presence of the mutation comprises performing droplet digital PGR (ddPCR). The digital PGR or ddPCR may be performed with at least one primer comprising any one of SEQ ID NOs: 1-14 or a portion thereof. The digital PGR or ddPCR may be performed with at least one probe comprising any one of SEQ ID NOs: 19-36. The digital PGR or ddPCR may be performed with probes comprising each of SEQ ID NOs: 19-36.

As stated hereinabove, the instant methods comprise determining the presence of a mutation (e.g., in histone H3, such as histone H3.3). In a particular embodiment, the method comprises determining the presence of a mutation in a gene(s) selected from the group of H3F3 A (e.g., p.K27M), HIST1H3B (e.g., p.K27M), HIST2H3C (e.g., p.K27M), ACVR1 (e.g., p.G328V/p.R206H), PPM1 (e.g., p.E525X), FK3R1 (e.g., p. 567E), PIK3CA (e.g., p.H1047R), IDH1 (e.g., p.R132H; isocitrate dehydrogenase (NADP(+)) 1, cytosolic (Gene ID:3417)) an

BRAF (e.g., p.V600E; B-Raf proto-oncogene, serine/threonine kinase (Gene ID: 673)) (e.g., 1, 2, 3, 4, 5, 6, 7, or all 8 genes are analyzed). In a particular embodiment, the gene(s) is selected from the group of H3F3A (e.g., p.K27M), HIST1H3B (e.g., p.K27M), ACVR1 (e.g.,

p.G328V/p.R2()6H), PPM ID (e.g., p.E525X), PIK3R1 (e.g., p. 567E), and PI 3CA (e.g., p.H1047R) (e.g., 1, 2, 3, or all 5 genes are analyzed). In a particular embodiment, the other gene(s) is selected from the group of HIST1H3B (e.g., p.K27M), ACVR1 (e.g.,

p.G328V/p.R206H), PPM1D (e.g., p.E525X), and PIK3R1 (e.g., p.K567E) (e.g.,2, 3, or all 4 genes are analyzed). In a particular embodiment, the mutation in histone H3 is K27M. In a particular embodiment, the histone H3 is H3F3A. In a particular embodiment, the method comprises determining the presence of a mutation in H3F3A and at least one other gene. A decrease in the presence of the mutations over time (e.g., when multiple biological samples are obtained after therapy) or compared to the amount observed prior to therapy, indicates that the cancer therapy administered to the subject is effective (e.g., inhibiting tumor growth).

OUgonucleotide probes are also provided with the instant invention. The probes are designed to have high affinity and specificity to the target site (e.g., the mutations set forth herein and, optionally, the wild- type gene). In a particular embodiment, oligonucleotide probe(s) target a gene (e.g., wild-type and/or mutant) selected from the group of H3F3A (e.g., p.K27M), HIST1H3B (e.g., p.K27M), HIST2H3C (e.g., p.K27M), ACVR1 (e.g., p.G328V/p.R206H), PPM1D (e.g., p.E525X), PIK3R1 (e.g., p.K567E), PIK3CA (e.g., p.H1047R), IDH1 (e.g., p.R132H) and BRAF (e.g., p.V600E).

The probes do not have an absolute requirement on length. However, the probes will typically be from about 10 to about 250 nucleotides, about 10 to about 100, about 10 about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 25 nucleotides, or about 10 to about 20 nucleotides. In a particular embodiment, the probe is at least about 10 nucleotides in length. The probe may be at least 85%, at least 90%, at least 95%, at least 97%, or, more preferably, 100% complementary to the target sequence. In a particular embodiment, the oligonucleotide probe is designed such that the mutation is towards the middle of the sequence of the probe (e.g., within the middle third of the probe length).

In a particular embodiment, the probe may comprise at least one nucleotide analog. For example, the nucleotide analogs may be used to increase annealing affinity and/or specificity and/or resistance to degradation. For example, the use of locked nucleic acid (LNA) bases within an oligonucleotide increases the affinity and specificity of annealing of the oligonucleotide to its target site (Kauppinen et al.(2005) Drug Discov. Today Tech., 2:287-290; Orum et al. (2004) Letters Peptide Sci., 10:325-334). Nucleotide analogs include, without limitation, nucleotides with phosphate modifications comprising one or more phosphorothioate, phosphorodithioate, phosphodiester, methyl phosphonate, phosphoramidate, methylphosphonate, phosphotriester, phosphoroaridate, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl substitutions; nucleotides with modified sugars; and nucleotide mimetics such as, without limitation, peptide nucleic acids (PNA), morpholino nucleic acids, cyclohexenyl nucleic acids, anhydrohexitol nucleic acids, glycol nucleic acid, threose nucleic acid, and locked nucleic acids (LNA). In a particular embodiment, the probes comprise at least one locked nucleic acid. The probes may comprise one of SEQ ID NOs: 19-36 or a sequence with at least 85%, at least 90%, at least 95%, or at least 97% identity to one of SEQ ID NOs: 19-36. In a particular embodiment, the probe comprises one of SEQ ID NOs: 19-36. The probes may comprise additional nucleotides 5' or 3' to the included SEQ ID NO. For example, the probe may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides 5' or 3' to the included SEQ ID NO. In a particular embodiment, the additional sequences are complementary to the target sequence.

The probes of the instant invention may comprise one or more fluorescent probes / fluorophores and/or quenchers. The fluorophores and or quenchers may be added to the 5' and or 3' termini of the probes. The fluorophores and/or quenchers may also be added to internal part of the probes (e.g., a ZEN probe). Fluorophores and/or quenchers are well known in the art (see, e.g., IDT, Coralville, IA). Examples of fluorophores and/or quenchers include, without limitation, 6-FAM, fluorescein, Cy3, Cy5, TAMRA, JOE, MAX, TET, Cy5.5, ROX, ATTO, TYE, Yakima Yellow®, HEX, TEX, Texas Red®, Iowa Black®, ZEN, and Alexa Fluor®. The fluorophores and/or quenchers used allow for the determination of the presence of the wild- type and/or mutant allele in a sample at the same time (see, e.g., the Examples). In a particular embodiment, the fluorophores and/or quenchers create an energy transfer pair (e.g., fluorescence resonance energy transfer (FRET)) (e.g.,as set forth in Table 1). In a particular embodiment, the probes comprise a fluorophore and/or quencher combination presented in Table 1. In a particular embodiment, the probe comprises any one of SEQ ID NOs: 19-36. In a particular embodiment, the probe comprises any one of SEQ ID NOs: 19-36 along with the modifications presented in Table 1.

Compositions comprising at least one probe of the instant invention are also provided. In a particular embodiment, the composition is an aqueous solution. In a particular embodiment, the composition comprises at least one probe comprising any one of SEQ ID NOs: 19-36. In a particular embodiment, the composition comprises individual probes comprising each of SEQ ID NOs: 19-36.

Compositions comprising at least one primer of the instant invention are also provided. In a particular embodiment, the composition is provided an aqueous solution. In a particular embodiment, the composition comprises at least one primer comprising any one of SEQ ID NOs: 1-18. In a particular embodiment, the composition comprises individual primers comprising each of SEQ ID NOs: 1-18.

Definitions

The following definitions are provided to facilitate an understanding of the present invention:

The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

"Pharmaceutically acceptable" indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

A "carrier" refers to, for example, a diluent, adjuvant, preservative (e.g.,Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., Tris HC1, acetate, phosphate), antimicrobial, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle (e.g., with which an active agent of the present invention is administered). Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of

Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington.

As used herein, the term "small molecule" refers to a substance or compound that has a relatively low molecular weight (e.g., less than 4,000, less than 2,000, particularly less than 1 kDa or 800 Da). Typically, small molecules are organic, but are not proteins, polypeptides, or nucleic acids, though they may be amino acids or dipeptides.

The term "treat" as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc. As used herein, the term "prevent" refers to the prophylactic treatment of a subject who is at risk of developing a condition resulting in a decrease in the probability that the subject will develop the condition.

As used herein, "diagnose" refers to detecting and identifying a disease or disorder in a subject. The term may also encompass assessing or evaluating the disease or disorder status (progression, regression, stabilization, response to treatment, etc.) in a patient known to have the disease or disorder.

As used herein, the term "prognosis" refers to providing information regarding the impact of the presence of a disease or disorder (e.g., as determined by the diagnostic methods of the present invention) on a subject's future health (e.g., expected morbidity or mortality, the likelihood of getting or risk of cholestasis). In other words, the term

"prognosis" refers to providing a prediction of the probable course and outcome of a disease/disorder and/or the likelihood of recovery from the disease/disorder.

As used herein, the term "subject" refers to an animal, particularly a mammal, particularly a human.

A "therapeutically effective amount" of a compound or a pharmaceutical

composition refers to an amount effective to prevent, inhibit, treat, or lessen the

symptoms of a particular disorder or disease. The treatment of a disease or disorder herein may refer to curing, relieving, and/or preventing the disease or disorder, the symptom(s) of it, or the predisposition towards it.

As used herein, the term "therapeutic agent" refers to a chemical compound or biological molecule including, without limitation, nucleic acids, peptides, proteins, and antibodies that can be u ed to treat a condition, disease, or disorder or reduce the symptoms of the condition, disease, or disorder.

The term "isolated" refers to the separation of a compound from other components present during its production or from its natural environment. "Isolated" is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not substantially interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, or the addition of stabilizers.

As used herein, a "biological sample" refers to a sample of biological material obtained from a subject, particularly a human subject, including a tissue, a tissue sample, cell(s), and a biological fluid (e.g., blood (e.g., whole blood), serum, plasma, urine, sweat, tears, saliva, mucosal secretions, sputum, CSF).

The term "probe" as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single- stranded or double- stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, the oligonucleotide probe typically contains about 10-250, about 10-100, about 10-50, about 15-30, about 15-25, or about 10-20 nucleotides. The probes herein may be selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to "specifically

hybridize" or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target, although they may. For example, a non-complementary nucleotide fragment may be attached to the 5' or 3' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non- complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically. A probe may be tagged or labeled (i.e., attached to an entity making it possible to identify a compound to which it is associated (e.g., fluorescent or radioactive tag). In certain embodiments, a label is selected from the group consisting of biotin, copper-DOTA, biotin-PEG3, aminooxyacetate, ¹⁹FB, ¹⁸FB, FITC-PEG₃, fluorescein and fluorescein derivatives (e.g., 5-carboxy fluorescein). In other embodiments, the label is selected from the group consisting of ⁶⁴Cu DOT A, ⁶⁸Ga DOT A, ¹⁸F, ^C , ⁶⁸Ga, ⁸⁹Zr, ⁱ²⁴I, ⁸⁶Y, ^94mTc, ^n0raXn, "C and ⁷⁶Br.

The term "primer" as used herein refers to an oligonucleotide, either RN A or DNA, either single- stranded or double-stranded, which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as appropriate temperature and H, the primer may be extended at its 3' terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product. The primer may vary in length depending on the particular conditions and

requirement of the application. For example, the oligonucleotide primer is typically about 10- 25 or more nucleotides in length, but can be significantly longer. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able to anneal with the desired template strand in a manner sufficient to provide the 3' hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template, though it may. For example, a non- complementary nucleotide sequence may be attached to the 5' end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.

The term "oligonucleotide," as used herein refers to sequences, primers and probes of the present invention, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.

The respective fluorophores are added to the 5' end of the probes, and the quenchers are added to the middle and at the 3' end of the probe. The symbol + following a nucleotide indicates a locked nucleic acid. IABkFQ: Iowa Black® FQ. SEQ ID NO is provided in parentheses. The SEQ ID NOs represent the nucleotide sequences without the probes or modifications.

Table 1: Primers & Probes for ddPCR

cDNA Mutation Forward Reverse Wild type Mutant

Gene

change (protein) Primer Primer probe probe

156-

5'- /5TET/TCGA

5'-CCAGACA FAM/TCGAG

ACCTCAGATA GAT+TTC+A

ACTGTTCAA AT+TTC+TC

BRAF 1799T>A V600E TATTTCTTCAT CT+GTAGCT

AC-3' T+GTAGCT/3

G-3' /3IABkFQ/

(18) IABkFQ/

(17) (35)

(36)

The following non- limiting examples are provided to illustrate various embodiments of the present invention. EXAMPLES

Example 1 : Methods

This example illustrates a sensitive and specific platform for discriminating rare, low abundant, tumor-associated circulating DNA in pediatric patients with mid-line glioma (MLG) tumors. Clinicopathological and genomic characteristics of the tumors of a pediatric MLG patient cohort are shown in Fig. 1A. Patients were diagnosed with MLGs harboring various mutation combinations, including oncohistone variants: 94% harbored histone 3 mutations (79% with H3.3K27M, 15% with H3.1K27M), and 6% were H3 wild type. The initial goal was to develop and validate a clinically relevant and minimally invasive Uquid biopsy platform suitable for detection and quantification of somatic mutations associated with pediatric MLGs. To address this, we validated our ddPCR probes for assessing mutation burden by using tumor genomic DNA from MLG patients harboring oncohistone H3F3A (p.K27M), HIST1H3B

(p.K27M) and partner mutants in genes ACVR1 (p.G328V, p.R206H), PPM1D (p.E525X), PIK3R1 (p.K567E), and BRAF (p.V600E) (FIGS. 2A-N). Additionally, no mutant clusters were observed where control brain genomic DNA was used as template (FIGS. 2A-N). To assess the sensitivity, we used a range (2.5ng, 250pg, 25pg and 2.5pg) of genomic DNA as template for ddPCR and detected a corresponding linear decrease (10 fold) in MAF, indicating a low (2.5pg) amount of DNA to be suitable for detection by ddPCR (FIGS. 3A-G).

To assess the specificity of our platform, we used a large cohort of control, non-CNS malignant specimens (CSF = 16; plasma = 20) and probed for wild type and mutant histone ctDNA. Our data determined that any allelic frequency detected as equal or below 0.001% to be false positive in the case of histone p.K27M analysis (Table 2). We also analyzed genomic DNA, CSF, and plasma obtained from DIPG patients who did not harbor histone 3 p.K27M mutations. No histone 117 mutant copies were detected in any of the specimen tested, indicating the specificity of our platform (FIGS. 4A-4B).

Example 2: CSF and plasma harbor circulating tumor DNA indicative of driver mutations associated with pediatric MLGs.

Liquid biome specimens were analyzed from 84 subjects (48 MLG patients, and 36 non- CNS diseased controls), enrolled in an ongoing clinical trial PNOC003 (NCT 227498), and consented for the Children's National (CN) brain tumor biorepository (FIG. IB). A cohort of 110 biofluid samples (30 CSF, 79 plasma, and 1 cyst fluid) representing 48 MLG patients was processed for liquid profiling (FIGS. 5A-B). CSF samples were collected at a single time point through the CN biorepository at pre-treatment, during therapy, and at postmortem from 27 MLG patients, while serial sampling at pre-treatment and postmortem was available for one patient with DIPG. Plasma specimens were obtained starting at diagnosis, and longitudinally throughout treatment from patients with DIPG enrolled in PNOC003 (NCT 2274987) (at initial biopsy n=23, during treatment n=15) (FIG. IB, Table 2). Three of the DIPG patients enrolled in PNOC003 also consented for CSF collection at postmortem via the CN biorepository. Two patients harbored H3 K27M and one was H3 wild type (FIG 1).

The results are shown in Table 2, which provides midline glioma patient demographic information, and samples analyzed for genomic and ctDNA studies. Genomic alterations were determined by sequencing conducted for PNOC003 clinical trial and biorepository sequencing study. Validation by ddPCR analysis of tumor samples is indicated in the table. EOT denotes end of therapy time point.

Histone 3 mutant and wild type alleles were detected in 75% of CSF specimens collected at diagnosis, 67% of those collected during treatment, and 90% of those collected at postmortem (Fig. 2a). H3 K27M- mutant ctDNA was detected in 89% of all CSF specimens analyzed from 27 MLGs, where 23 of these were confirmed to harbor oncohistones as assessed by tumor DNA analysis.

We have previously shown that in addition to oncohistone mutations, MLGs harbor obligate partner mutations in genes such as ACVR1, TP53, PPM ID, and PIK3R1. We thus sought to determine whether biofluids represent a source for detection of multiple mutations for a particular patient. We first used tumor DNA known to harbor oncohistone and partner mutations (ACVR1 p.R206H, PIK3R1 p.K567E, or BRAF p.V600E) to optimize the experimental and analytical approach for multiplexed assays (FIGS. 7A-F). We then tested CSF obtained from these patients and showed the feasibility of detecting mutant and wild type alleles, for oncohistone and obligate partners in ACVR1, PIK3R1 or BRAF (FIGS. 7A-F). Given the paucity of tumor DNA, and the invasive nature of surgical biopsy, our multiplexed mutation analysis allows for maximized clinical utility of a single accessible biofluid specimen.

We then assessed the effect of CSF location on ctDNA abundance. CSF collected from lateral ventricles, lumbar spine, cervical spine, subdural (at biopsy), ventriculoperitoneal (VP) shunt, and the fourth ventricle (cisternae magna tap) was analyzed by ddPCR. While we detected a significantly higher mutation allelic frequency in CSF collected from adjacent locations compared to distant, we report successful detection of H3 K27M ctDNA in all locations tested (FIGS. 8A-C). We previously reported the suitability of the cyst fluid for identification of tumor associated proteins. Here, we analyzed tumor cyst fluid, CSF, and tumor genomic DNA obtained at postmortem from a patient diagnosed with DIPG. We showed that the cyst fluid is highly enriched for tumor DNA as suggested by the high number of histone mutant DNA copies (FIGS. 9A-D).

To assess the enrichment of ctDNA in CSF versus plasma, we tested these two biofluids and found that in general, a significantly higher allelic frequency of ctDNA was detected in CSF (n=27 patients, 30 specimens) compared to plasma (n=20, 77 specimens) (p < 0.0001), which may indicate ctDNA enrichment in CSF due to proximity to the tumor mass (FIG. 6B). This cohort-based result was confirmed by our matched analysis (n=2), where we found an enrichment of ctDNA in CSF compared to plasma within the same patient (FIG. 6C). Our data indicate that both CSF and plasma provide a suitable medium for detection of ctDNA, and demonstrates the potential of liquid biopsy for identification of tumor-associated somatic mutations and replacing, or complementing current invasive and costly surgical biopsies.

Example 3: Assessment of treatment response using ctDNA quantification.

To investigate the clinical translation of liquid biopsy, we studied plasma obtained from patients diagnosed with DIPG who enrolled in an ongoing clinical trial (PNOC 003, NCT 2274987). Analysis of upfront and serial plasma samples probing for histone mutations (H3.3 and H3.1) indicated the following: i) histone mutations were successfully detected at diagnosis in 80% (n=16 of 20) of patients harboring known histone mutations determined by genomic analysis of pretreatment biopsies; ii) histone mutation was not detected at diagnosis in 20% of patients with histone mutations (n=4 of 20), hi) however, among these four patients, histone mutation was subsequently detected at post-radiation in two patients where serial samples were available (FIGS. 6D-E). MR image analysis of the two patients for whom ctDNA was absent at diagnosis, but present post-radiation indicated an increase in tumor volume following radiation (patient 12: 40.6mm³ to 62.4mm³, and patient 21: 28.6mm³ to 34.8mm³) (FIG. 6F), suggesting that changes in tumor architecture in response to radiation, or temporary disruption of the blood brain barrier (BBB) may contribute to the availability of ctDNA in plasma at different time points. Similarly, our analysis of serially collected CSF from a patient with H3 K27M DIPG at pre-treatment and at postmortem, revealed an increase in ctDNA abundance at the later stage of disease (FIGS. 10A- B).

To assess tumor response to treatment, longitudinal plasma samples obtained from enrolled patients (PNOC 003, NCT 2274987) at diagnosis, during treatment, and at tumor growth were studied (n=15). We found that in general, ctDNA abundances varied throughout the course of the treatment (see FIG.l 1). Importantly, patient- specific temporal analysis of changes in musculoaponeurotic fibrosarcoma oncogene (MAF) histone 3 mutation indicated a close association of ctDNA abundance with both clinical course of the disease, and tumor response as indicated by MR imaging (FIGS. 13A-D). Specifically, longitudinal ctDNA analysis of one patient showed a decrease in histone MAF post- radiation, stabilization during the first treatment course (panobinostat and mebendazole), a substantial increase at tumor growth, and a subsequent drop after patient underwent the second treatment course (FIG. 1 IB). To establish association of ctDNA MAF with tumor volume, a central MRI review was conducted to compare tumor volume (mm³) throughout the treatment course of patients enrolled in the clinical trial (PNOC 003, NCT 2274987) (FIG. 1 IB). In the nine patients who followed PNOC003 recommended therapy and had serial plasma samples collected throughout treatment, ctDNA abundance agreed with tumor volume reduction as assessed by MR imaging (FIG.11C).

To further compare ctDNA-based MAF with MR imaging, we performed statistical analysis of MAF (H3 ctDNA) at biopsy and post-radiation as well as tumor volume assessments using MR images. Our data showed that in 15 patients ctDNA MAF significantly decreased post- radiation when assessed by both liquid biopsy (p=0.004) and MR imaging (p=0.01) (Fig. 3d). Plasma ctDNA-MRI relationship was further compared by monitoring tumor response as assessed by MAF and MRI data at diagnosis and post-treatment (pre-cycle 3). Although both approaches indicated response, the MAF and tumor seize decrease were not significant (p=0.06 for decrease in ctDNA MAF and tumor volume measurements) (FIG. 1 IE).

We then assessed whether an increase in H3 K27M ctDNA accompanied tumor spread as determined by MRI, molecular, and/or histological studies. Tumor extension was determined by MRI obtained during the course of the treatment, or molecular and histopathological review of autopsied brain specimens. Tumor involvement beyond pons for patients enrolled in the clinical trial (PNOC 003, NCT 2274987) was determined by central MRI review at diagnosis, during therapy, or at tumor growth. We found that increased levels of CSF ctDNA corresponded to tumor spread; however, the small sample size for the availability of CSF for DIPGs with tumor dissemination precluded statistical significance. On the contrary, plasma collected at various times throughout disease (diagnosis, during treatment, or at tumor growth) was not predictive or indicative of tumor spread beyond pons (FIG. 1).

Outcomes for children diagnosed with MLG have not changed despite decades of clinical research. Recent advancements in genomic, epigenomic, and proteomic profiling, however, provide new opportunities for accelerating development of novel therapeutic interventions for childhood cancers. Collection of postmortem specimens resulted in the identification of oncohistones as driver mutations as well as other partner mutations, and newer clinical trials are building upon this knowledge to identify rational, biologically targeted treatment approaches.³' ⁴' ²¹ However, limited availability of biopsy in the pons and difficulty in accurately assessing patients' responses to therapy are current challenges in pediatric MLGs.

Liquid biopsy is an emerging tool for diagnosing, and measuring efficacy of treatment in adult cancer patients. While molecular profiling of tumors is a localized method, a liquid biopsy approach provides a systemic molecular overview. ctDNA has been used to determine patient's mutation profiles, as a biomarker for molecular-based disease monitoring, and recurrence in adult chronic lymphocytic leukemia, breast, and colon cancer.¹³'¹⁶'²³ The only previously reported liquid biopsy approach for pediatric MLGs was established using Sanger sequencing.¹⁹ The disclosure herein for the detection and quantification of tumor-associated circulating DNA using ddPCR allows for rapid, more sensitive, far less costly and less invasive method for surveying tumor mutations, and represents a key advance particularly for tumors with limited tissue acquisition or prohibitive sampling at multiple time points.

While we report the detection of ctDNA in both CSF and plasma, the exact mechanism of tumor DNA release into the biofluid is not well understood. A survey of published manuscripts indicated that ctDNA is released from multiple sources, including living cells, apoptotic and/or necrotic tumor cells. We also found that CSF yielded a significantly higher amount of ctDNA compared to plasma. This is most likely due to the location of biofluids in relation to site of tumor, and challenges of overcoming the BBB for release of ctDNA into plasma.

Our lack of detection of ctDNA in plasma obtained at biopsy from four patients (H3 mutant by biopsy) may suggest high BBB integrity and thus lack of circulating tumor DNA. Subsequent analyses detected ctDNA in plasma drawn for two of these patients at post radiation, indicating the potential role of radiation therapy (RT) for temporarily disrupting BBB; hence, resulting in detection of ctDNA. Moreover, as the cellular turnover in a growing tumor increases, apoptotic and necrotic tumor cells are also responsible for the increased release of ctDNA into biofluids. As such, we expected to observe an initial spike in ctDNA as tumor cells die and release DNA into biofluids. However, our tested time points fell after the completion of RT when the tumor mass was reduced in most patients as assessed by MRI, corresponding to the expectant reduced number of ctDNA. An issue to be determined is whether inclusion of more time points during the course of RT, would detect an initial spike in ctDNA immediately after the first radiation treatment, followed by stabilization of ctDNA at maximal tumor response. Our study suggests that collection of multiple plasma draws at diagnosis and during treatment will be more informative as ctDNA may not be detectable in initial blood draws.

We have recently shown that DIPG tumor cells disseminate throughout the brain during the course of disease. Our ctDNA analysis in DIPG patients was indicative of tumor expansion beyond pons, where an increased amount of ctDNA in CSF was observed in patients who exhibited tumor spread. Studies of a larger cohort in clinical settings are required to assess the statistical significance of our finding. More importantly, our results indicate the unique strength of liquid biopsy for assessing the molecular landscape of MLGs, and potential for longitudinal assessment of tumor response to therapy, which is a new tool that is complementary to MR imaging. Similar to the clinical utility of ctDNA for monitoring response to therapy with respect to MRIs in adult GBMs, despite a small sample size, we found significant reduction in ctDNA following RT. Similar patterns in temporal changes of ctDNA and MRI assessments of tumor response indicated that our ddPCR analysis of plasma is highly translational, and offers a novel platform for assessing tumor response, regression and/or progression in MLGs. We provide a valuable tool that may be incorporated into future clinical trials for longitudinal assessment of treatment related molecular changes. Our liquid biopsy approach may also be expanded for facilitating diagnosis, longitudinal monitoring, and assessing recurrence in other childhood CNS cancers. In summary, we have shown that CSF and plasma ctDNA analysis of children with MLG is feasible, shows promise for detecting mutational load, provides an additional means for disease characterization, and, more importantly is a clinically relevant and sensitive method for assessing tumor response to treatment.

Table 2: Assessing specificity of a ddPCR platform by testing non-CNS malignant pediatric CSF and plasma.

Non-CNS

Diseased

Biofluid

Plasma Gene Mutation Average MAF type

Patient

ID

1252 Plasma H3F3A P.K27M 0.000%

1253 Plasma H3F3A P.K27M 0.000%

1255 Plasma H3F3A P.K27M 0.000%

1 140 Plasma HIST1H3B P.K27M 0.000%

1 141 Plasma HIST1H3B P.K27M 0.000%

1 142 Plasma HIST1H3B P.K27M 0.000%

1 143 Plasma HIST1H3B P.K27M 0.000%

1 144 Plasma HIST1H3B P.K27M 0.000%

1 145 Plasma HIST1H3B P.K27M 0.000%

1 146 Plasma HIST1H3B P.K27M 0.000%

1 147 Plasma HIST1H3B P.K27M 0.000%

1 148 Plasma HIST1H3B P.K27M 0.000%

942 CSF H3F3A P.K27M 0.000%

943 CSF H3F3A P.K27M 0.000%

945 CSF H3F3A P.K27M 0.000%

946 CSF H3F3A P.K27M 0.000%

947 CSF H3F3A P.K27M 0.000%

951 CSF H3F3A P.K27M 0.000%

955 CSF H3F3A P.K27M 0.001%

956 CSF H3F3A P.K27M 0.000%

853 CSF H3F3A P.K27M 0.000%

862 CSF H3F3A P.K27M 0.000%

856 CSF H3F3A P.K27M 0.000%

1 187 CSF H3F3A P.K27M 0.000%

1 195 CSF H3F3A P.K27M 0.000%

1 196 CSF H3F3A P.K27M 0.000%

1203 CSF H3F3A P.K27M 0.000%

1205 CSF H3F3A P.K27M 0.000%

943 CSF HIST1H3 P.K27M 0.000%

B

945 CSF HIST1H3 P.K27M 0.000%

B

946 CSF HIST1H3 P.K27M 0.000%

B Table 2: Assessing specificity of a ddPCR platform by testing non-CNS malignant pediatric CSF and plasma.

Non-CNS

Diseased

Biofluid

Plasma Gene Mutation Average MAF

type

Patient

ID

947 CSF HIST1H3 P.K27M 0.000%

B

951 CSF HIST1H3 P.K27M 0.000%

B

955 CSF HIST1H3 Ρ.Κ27Μ 0.001 %

B

956 CSF HIST1H3 Ρ.Κ27Μ 0.000%

B

853 CSF HIST1H3 Ρ.Κ27Μ 0.000%

B

862 CSF HIST1H3 Ρ.Κ27Μ 0.000%

B

856 CSF HIST1H3 Ρ.Κ27Μ 0.000%

B

1 187 CSF HIST1H3 Ρ.Κ27Μ 0.000%

B

Mutation allele frequencies (MAFs) for H3F3A p.K27M and HIST1H3B p.K27M in plasma and CSF collected from pediatric non-CNS diseased controls indicating the false positive rate of detection. Average MAF values for non- CNS diseased plasma analyzed for H3F3A p.K27M mutation represent technical triplicates, all other average MAF values for plasma and CSF analyzed for H3F3A and HIST1H3B p.K27M represent technical duplicates.

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Claims

WHAT IS CLAIMED:

1. A method of determining the efficacy of a cancer therapy, said method comprising: a) administering a cancer therapy to a subject in need thereof;

b) obtaining a biological sample from the subject after administration of the cancer therapy to the subject; and

c) deteraiining the presence of a mutation in one or more genes selected from the group consisting of H3F3A, HIST1H3B, HIST1H3C, HIST2H3C, ACVRl, PPMID, PIK3R1, PIK3CA, IDH1, and BRAF in the biological sample,

wherein a decrease in the presence of the mutation(s) compared to the amount observed prior to administration of the cancer therapy indicates that the cancer therapy administered to the subject is effective.

2. The method of claim 1, wherein steps b) and c) are repeated at least once, wherein a decrease in the presence of the mutation(s) over time indicates that the cancer therapy administered to the subject is effective.

3. The method of claim 1, wherein said biological sample is a fluid.

4. The method of claim 3, wherein said biological fluid is blood, plasma, serum, CSF, or urine.

5. The method of claim 3, wherein said biological fluid is blood or plasma.

6. The method of claim 1 , wherein said cancer is a brain tumor.

7. The method of claim 6, wherein said cancer is a pediatric brain tumor.

8. The method of claim 6, wherein said brain tumor is a diffuse glioma or a diffuse intrinsic pontine glioma (DIPG).

9. The method of claim 1, wherein step c) comprises performing a method selected from the group consisting of quantitative polymerase chain reaction (qPCR), quantitative real-time polymerase chain reaction (qRTPCR), digital droplet PCR, (ddPCR), sequencing, northern blotting, and Southern blotting.

10. The method of claim 1, wherein step c) comprises determining the presence of a mutation in H3F3A.

11. The method of claim 10, wherein step c) comprises determining the presence of a K27M mutation in H3F3A.

12. The method of claim 11, wherein step c) further comprises determining the presence of a mutation in at least one other gene selected from the group consisting of HIST1H3B, HIST1H3C, HIST2H3C, ACVRl, PPM ID, PIK3R1, PIK3CA, IDH1, and BRAF.

13. The method of claim 11, wherein step c) further comprises determining the presence of a mutation in at least one other gene selected from the group consisting of HIST1H3B, ACVRl, PPM1D, PIK3R1, and PIK3CA.

14. The method of claim 11, wherein step c) further comprises determining the presence of a mutation in at least one other gene selected from the group consisting of HIST1H3B, ACVRl, PPMlD, and PIK3Rl.

15. A method of treating cancer in a subject in need thereof, said method comprising:

a) determining the presence of a mutation in one or more genes selected from the group consisting of H3F3A, HIST1H3B, HIST1H3C, HIST2H3C, ACVRl, PPM ID, PIK3R1, PIK3CA, IDH1, and BRAF in a biological sample, and

b) administering a cancer therapy to said subject if the mutation(s) is present.

16. The method of claim 15, wherein said biological sample is a fluid.

17. The method of claim 16, wherein said biological fluid is blood, plasma, serum, CSF, or urine.

18. The method of claim 16, wherein said biological fluid is blood or plasma.

19. The method of claim 15, wherein said cancer is a brain tumor.

20. The method of claim 19, wherein said cancer is a pediatric brain tumor.

21. The method of claim 19, wherein said brain tumor is a diffuse glioma or a diffuse intrinsic pontine glioma (DIPG).

22. The method of claim 15, wherein step a) comprises performing digital PCR or droplet digital PCR (ddPCR).

23. The method of claim 15, wherein step a) comprises determining the presence of a mutation in H3F3A.

24. The method of claim 22, wherein step a) comprises determining the presence of the K27M mutation in H3F3A.

25. The method of claim 23, wherein step a) further comprises determining the presence of a mutation in at least one other gene selected from the group consisting of HIST1H3B, HIST1H3C, HIST2H3C, ACVRl, PPM ID, PIK3R1, PIK3CA, IDH1, and BRAF.

26. The method of claim 23, wherein step a) further comprises determining the presence of a mutation in at least one other gene selected from the group consisting of HIST1H3B, ACVRl, PPM1D, PIK3R1, and PIK3CA.

27. The method of claim 23, wherein step a) further comprises determining the presence of a mutation in at least one other gene selected from the group consisting of HIST1H3B, ACVRl, PPMID, and PIK3R1.

28. A method of detecting a mutation in one or more genes selected from the group consisting of H3F3A, HIST1H3B, HIST1H3C, HIST2H3C, ACVRl, PPMID, PIK3R1, PIK3CA, IDHl, and BRAF in a blood or plasma sample from a human subject between 1 and 18 years of age, said method comprising performing digital PCR or droplet digital PCR (ddPCR) on the blood or plasma sample to determine the presence of a mutation in one or more genes selected from the group consisting of H3F3A, HIST1H3B, HIST1H3C, HIST2H3C, ACVRl, PPMID, PIK3R1, PIK3CA, IDHl, and BRAF in a biological sample.

29. The method of claim 28, wherein the subject has cancer.

30. The method of claim 29, wherein the cancer is a brain tumor.

31. The method of claim 30, wherein said brain tumor is a diffuse glioma or a diffuse intrinsic pontine glioma (DIPG).

32. The method of claim 28, wherein the one or more genes comprise H3F3A.

33. The method of claim 32, wherein the mutation is a K27M mutation in H3F3A.

34. The method of claim 33, further comprising determining the presence of a mutation in at least one other gene selected from the group consisting of HIST1H3B, HIST1H3C, HIST2H3C,

ACVRl, PPMID, PIK3R1, PIK3CA, IDHl, and BRAF.

35. The method of claim 33, further comprising determining the presence of a mutation in at least one other gene selected from the group consisting of HIST1H3B, ACVRl, PPMID, PIK3R1, and PIK3CA.

36. The method of claim 33, wherein step c) further comprises determining the presence of a mutation in at least one other gene selected from the group consisting of HIST1H3B, ACVRl, PPMW, and PIK3Rl.

37. An oligonucleotide probe comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 19-36, wherein the probe further comprises a label.

38. A composition comprising at least one oligonucleotide probe of claim 37.